Name: three-dimensional culture of vascularized thermogenic adipose tissue from microvascular fragments
Uploaded: 2023-02-03T13:30:00
Description: Watch this Scientific Journal Video about Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments at JoVE.com

Dr. Acosta is supported by the National Institutes of Health grants CA148724 and TL1TR002647. Dr. Gonzalez Porras is supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health, under Award Number F32-0DK122754. This work was supported, in part, by the National Institutes of Health (5SC1DK122578) and the University of Texas at San Antonio Department of Biomedical Engineering. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Figures were partially created with Biorender.com.

This study was conducted in compliance with the Animal Welfare Act and the Implementing Animal Welfare Regulations in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Texas at San Antonio.
NOTE: For the steps described below, male Lewis Rats are utilized. Slight protocol adjustments must be made for a female, as well as mouse microvascular fragment ...

The field of brown/beige adipose tissue engineering is largely immature22,23,24,25,26,27,28, with the bulk of adipose models being developed for white adipose tissue8,22,31. Engineered brown/beige m...

The goal of this protocol is to describe an approach for developing vascularized beige adipose tissue from a single, potentially autologous source, microvascular fragment (MVF). Brown and beige adipose tissues have been demonstrated to display beneficial properties related to metabolic regulation; however, the small volume of these adipose tissue depots in adults limits the potential impact on systemic metabolism, particularly in diseased conditions such as obesity or type 2 diabetes1,2,3,4,5,6,7. There is significant interest in brown/beige fat as a therapeutic target for preventing the harmful metabolic effects linked with obesity and its comorbidities8,9,10,11,12.
MVFs are vessel structures that can be directly isolated from adipose tissue, cultured, and maintained in a three-dimensional configuration for extended periods of time13,14,15. Previous work from our group, and others, have begun to exploit the multicellular and multipotent capacity of MVFs, specifically as it relates to adipose tissue formation16,17,18. As a buildup of this work, we recently demonstrated that MVFs derived from rodent models of healthy and type 2 diabetes19 and from human subjects (adults over 50 years of age)20 contained cells capable of being induced to form thermogenic, or beige, adipose tissue.
Herein is an innovative approach from which a single source MVF is utilized, not only capable of creating beige adipose tissue but also its associated and critical vascular component21. The use of this technique could be of great value for studies looking for a straightforward tissue-engineered approach for thermogenic adipose tissue formation. Unlike other methods aspiring to engineer beige adipose tissue22,23,24,25,26,27,28, the process described in this study does not require using multiple cell types or complex induction regimens. Vascularized beige and white fat models can be created with MVFs originating from rodent and human sources, demonstrating great translation potential. The end product of this protocol is an engineered beige thermogenic fat tissue with a structure and metabolic function comparable to brown adipose tissue. Overall, this protocol presents the idea that an easily accessible and possibly autologous source MVF may be a worthwhile therapeutic intervention and tool for studying metabolic disorders.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aminocaproic Acid</td>
			<td>Sigma Aldrich</td>
			<td>A2504-100G</td>
			<td>Added in DMEM at the concentration of 1 mg/mL</td>
		</tr>
		<tr>
			<td>Blunt-Tipped Scissors</td>
			<td>Fisher scientific</td>
			<td>12-000-172</td>
			<td>Sterilize in autoclave</td>
		</tr>
		<tr>
			<td>Bovin Serum Albumin (BSA)</td>
			<td>Millipore</td>
			<td>126575-10GM</td>
			<td>Diluted in PBS to 4 mg/mL and 1 mg/mL</td>
		</tr>
		<tr>
			<td>Collagenase Type 1</td>
			<td>Fisher scientific</td>
			<td>NC9633623</td>
			<td>Diluted to 6 mg/mL in BSA 4 mg/mL, Digestion of minced fat</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Thermo Scientific</td>
			<td>AC230302500</td>
			<td>Diluted in ethanol at a 2 mg/ml stock concentration</td>
		</tr>
		<tr>
			<td>Disposable underpads</td>
			<td>Fisher scientific</td>
			<td>23-666-062</td>
			<td>For fluid absorption during surgery</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Fisher scientific</td>
			<td>08-951-5</td>
			<td>Sterilize in autoclave</td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium (DMEM)</td>
			<td>Fisher scientific</td>
			<td>11885092</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium/Nutrient Mixture F-12 Ham (DMEM/F12)</td>
			<td>Sigma Aldrich</td>
			<td>D8062</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Fisher scientific</td>
			<td>16140089</td>
			<td>Added in DMEM to 20% v/v.</td>
		</tr>
		<tr>
			<td>Fibrinogen&#160;</td>
			<td>Sigma Aldrich</td>
			<td>F8630-25G</td>
			<td>Solubilized in DMEM at the concentration of 20 mg/mL, Protein found in blood plasma and main component of hydrogel</td>
		</tr>
		<tr>
			<td>Flask, 250 mL</td>
			<td>Fisher scientific</td>
			<td>FB500250</td>
			<td>Allows for digestion of fat using a large surface area</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher scientific</td>
			<td>50-264-21</td>
			<td>Sterilize in autoclave, For handling of tissue and filters</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma Aldrich</td>
			<td>F6886</td>
			<td>Diluted in ethanol at a 10 mM stock concentration</td>
		</tr>
		<tr>
			<td>Human MVF</td>
			<td>Advanced Solutions Life Scienes, LLC</td>
			<td>https://www.advancedsolutions.com/microvessels</td>
			<td>Human MVFs (hMVFs) isolated from three different patients (52-, 54-, and 56-year old females) were used in the current study.&#160;</td>
		</tr>
		<tr>
			<td>Indomethacine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>I7378</td>
			<td>Diluted in ethanol at a 12.5 mM stock concentration</td>
		</tr>
		<tr>
			<td>Insulin from porcine pancreas</td>
			<td>Sigma Aldrich</td>
			<td>I5523</td>
			<td>Diluted in 0.01 N HCl at a 5 mg/ml stock concentration</td>
		</tr>
		<tr>
			<td>MycoZap</td>
			<td>Fisher scientific</td>
			<td>NC9023832</td>
			<td>Added in DMEM to 0.2% w/v, Mycoplasma Prophylactic&#160;</td>
		</tr>
		<tr>
			<td>Pennycilin/Streptomycin (10,000 U/mL)</td>
			<td>Fisher scientific</td>
			<td>15140122</td>
			<td>Added in DMEM to 1% v/v.</td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene (100 mm x 15 mm).</td>
			<td>Fisher scientific</td>
			<td>351029</td>
			<td>3 for removal of blood vessels and mincing, 8 (lid) for presoaking of screens &#38; 8 (dish) for use when filtering with 500 or 37 &#181;M screens</td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene (35 mm x 10 mm).</td>
			<td>Fisher scientific</td>
			<td>50-202-036</td>
			<td>For counting fragments</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS)</td>
			<td>Fisher scientific</td>
			<td>14-190-250</td>
			<td>Diluted to 1x with sterile deionized water.</td>
		</tr>
		<tr>
			<td>Rat Clippers (Andwin Mini Arco Pet Trimmer)</td>
			<td>Fisher scientific</td>
			<td>NC0854141</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Fisher scientific</td>
			<td>R0106200MG</td>
			<td>Diluted in DMSO at a 10 mM stock concentration</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14059-11</td>
			<td>1 for initial incision, 1 for epididymal incision, 1 for tip clipping</td>
		</tr>
		<tr>
			<td>Screen&#160; 37 &#181;M&#160;</td>
			<td>Carolina Biological Supply Company</td>
			<td>652222R</td>
			<td>Cut into 3&#34; rounded squares and sterilized in ethylene oxide, Fragment entrapment and removal of very small fragments/single cells and debris</td>
		</tr>
		<tr>
			<td>Screen 500 &#181;M&#160;</td>
			<td>Carolina Biological Supply Company</td>
			<td>652222F</td>
			<td>Cut into 3&#34; rounded squares and sterilized in ethylene oxide, Removes larger fragments/debris</td>
		</tr>
		<tr>
			<td>Serrated Hemostat</td>
			<td>Fisher scientific</td>
			<td>12-000-171</td>
			<td>Sterilize in autoclave, For clamping of skin before incision</td>
		</tr>
		<tr>
			<td>Steriflip Filter 0.22 &#956;m&#160;</td>
			<td>Millipore</td>
			<td>SE1M179M6</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Fisher scientific</td>
			<td>6051601KU</td>
			<td>Diluted in deionzed water to 10 U/mL, Used as a clotting agent turning fibrinogen to fibrin</td>
		</tr>
		<tr>
			<td>Thyroid hormone (T3)</td>
			<td>Sigma Aldrich</td>
			<td>T2877</td>
			<td>Diluted in 1N NaOH at a 0.02 mM stock concentration</td>
		</tr>
		<tr>
			<td>Zucker diabetic fatty (ZDF) rats - obese (FA/FA) or lean (FA/+) male&#160;</td>
			<td>Charles River</td>
			<td>https://www.criver.com/products-services/find-model/zdf-rat-lean-fa?region=3611 
			https://www.criver.com/products-services/find-model/zdf-rat-obese?region=3611</td>
			<td>Obtained from Charles River (Wilmington, MA). Rats were acquired at 4 weeks of age and fed Purina 5008 until euthanasia (15-19 weeks of age). Glucose levels (blood from the lateral saphenous vein) were greater than 300 mg/dL in all FA/FA rats used in the study. All animals were housed in a temperature-controlled environment with a 12-h light-dark cycle and fed ad libitum.</td>
		</tr>
	</tbody>
</table>

three-dimensional culture of vascularized thermogenic adipose tissue from microvascular fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

There are a few key phenotypic morphological characteristics of beige/brown adipose tissue: it is multilocular/contains small lipid droplets, possesses a large number of mitochondria (the reason for its characteristically &#34;brownish&#34; appearance in vivo), correspondingly has a high oxygen consumption rate/mitochondrial bioenergetics, is highly vascularized, has increased lipolysis/insulin-stimulated glucose uptake, and, most notoriously, expresses high levels of uncoupling protein 1 (UCP1), a mitochondrial...

Watch this Scientific Journal Video about Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments at JoVE.com

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

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